T cells play a vital role in the adaptive immune system's ability to combat invading pathogens and establish defenses against various diseases. T-cell receptors (TCRs) form the basis of specificity for each individual T cell. To advance discovery in pathogenesis, host response, and potential therapies for cancer, infectious diseases, and autoimmune disorders, we need powerful tools that can decode TCR sequences from both TCR alpha and beta chains in mouse samples.
Our new SMART-Seq Mouse TCR (with UMIs), referred to as the “mTCRv2” kit, is designed to address this need. We have improved the chemistry to ensure the new kit has:
Simple methodology—with a streamlined protocol for pooling
Applications of mTCRv2 include but are not limited to researching:
The development of lymphocytes—how different factors drive compartmentalization, differentiation, and distribution of T cells in different mouse tissues
Biomarkers in mouse disease models—how TCR signatures function as biomarkers for prognosis, diagnosis, and treatment of autoimmune diseases like Type 1 diabetes and rheumatoid arthritis
Responses to cancer immunotherapy—how relative abundance of different T-cell clones affects sensitivity to cancer immunotherapy
Host response in infectious diseases—how T cells influence the responsiveness to antiretroviral therapy or cause remission in certain cases
A SMART-based approach for sensitive and unbiased detection of TCR transcripts
Exactly how does mTCRv2 technology work? First-strand cDNA synthesis is dT-primed and results in the addition of non-templated nucleotides. The TCR SMART UMI Oligo, which has UMI sequences incorporated, then anneals to the non-templated oligonucleotides added by the reverse transcriptase. These oligonucleotides serve as a new template for primers to bind during PCR1 to amplify the entire variable region and considerable portion of the constant region from both TCR alpha and beta chains (Figure 1, Panel A). Products from PCR1 are used as templates for the second round of semi-nested PCR, in which the entire variable region gets amplified and indexed with the UDI sets. Following bead purification, size selection, and quality check, the library is ready for Illumina sequencing on various Illumina sequencers (Figure 1, Panel B).
Conduct innovative experiments using a wide range of sample quantities and types
To evaluate the compatibility of mTCRv2 with different input amounts, we prepared libraries from 10, 100, 250, 500, and 1,000 ng of mouse spleen RNA. Clonotype counts from both alpha and beta chains increase consistently with higher input amount (Figure 2, Panel A).
Meanwhile, we tested the performance of the new mTCRv2 kit across different sample types. Libraries were prepared from 200 ng of RNA isolated from four sample types frequently used in mouse studies—thymus, bone marrow, whole blood, and spleen. The sequencing results indicate that TCR sequences are captured with high sensitivity in all these sample types, suggesting the mTCRv2 kit is well suited for the analysis of mouse TCRs (Figure 2, Panel B).
Study the mouse TCR landscape with accuracy and reproducibility
Our mTCRv2 kit not only allows for sensitive detection of TCR sequences in different sample types; it also guarantees accuracy and reproducibility with the incorporation of UMIs. We prepared TCR libraries using mouse spleen RNA and examined how clonotype counts changed with increasing sequencing depth. Incorporation of UMIs allowed us to remove TCR sequences incorrectly called due to PCR duplicates and sequencing errors, which is crucial in clinical research (Figure 3, Panel A). UMIs also ensure confident clonotype identification at a lower sequencing depth. With fewer reads required to identify the same number of clonotypes, customers can free up the extra sequencing reads available to pool more samples together.
When looking for critical biomarkers such as rare TCRs, reproducibility is essential. To evaluate the reproducibility of mTCRv2, we prepared TCR libraries from 10 ng and 100 ng of mouse spleen RNA using the mTCRv2 protocol. Technical replicates for each input amount were sequenced on a MiSeq® sequencer with 300-cycle and 600-cycle cartridges to cover the CDR3 region and full-length V(D)J sequences, respectively. The Venn diagram indicates at least 87% clonotype overlap (Figure 3, Panel B), suggesting outstanding reproducibility.
Save on costs with library pooling and flexible sequencing options
We understand the increasing need for high-throughput analysis in basic and clinical research. We designed the mTCRv2 kit to be compatible with our current UDI sets, enabling you to pool up to 384 TCR libraries and to sequence on Illumina platforms with patterned flow cells. Sequencing the libraries with UDIs helps prevent issues like index hopping, ensuring high data quality and maximum discovery.
Besides saving sequencing costs by pooling samples with UDIs, the kit also allows the flexibility to sequence mouse TCR libraries in different ways. Sequence either the entire full-length V(D)J with 300-cycle, paired-end sequencing or just sequence the CDR3 region with a shorter sequencing length. This allows you to decode the most important sequences underlying T cell-antigen interaction while staying within budget (Figure 4).
Bulk sample mouse TCR profiling is important in advancing our understanding of immune responses in mouse models, allowing us to form better strategies for diagnosis and treatment. SMART-Seq Mouse TCR (with UMIs) allows you to discover critical biomarkers within mouse TCR sequences for the development of better immunotherapies and vaccine strategies. Along with the updated Cogent NGS Immune Profiler software, you can enjoy a complete solution to support your mouse study, from library prep to data analysis.
Materials and methods
Mouse spleen RNA was obtained internally (Takara Bio, Cat. #636605). Mouse Whole Blood (Cat. #MR-705-C57), Bone Marrow (Cat. #MR-704-C57), and Thymus (Cat #MR-702-C57) RNA were purchased from AMSBIO. All RNA samples were quantified using the ThermoFisher Nanodrop and diluted as necessary to achieve the range of RNA inputs tested (1 ng–1 μg).
Library purification, quantification, and sequencing
All libraries containing TRA and TRB sequences were generated using the SMART-Seq Mouse TCR (with UMIs) kit as per the user manual. Following purification and size selection, libraries were quantified using the Qubit and the Agilent 2100 Bioanalyzer. Libraries were sequenced on either an Illumina MiSeq platform with 600-cycle V3 cartridges (Illumina, Cat. #MS-102-3003), Illumina MiSeq platform with 300-cycle V3 cartridges (Illumina, Cat. #MS-102-3001), NextSeq 550 platform with 300-cycle Mid Output cartridges (Illumina, Cat. #20024905), or MiniSeq with 300-cycle Mid Output cartridges (Illumina, Cat. #FC-420-1004). Sequencing data analysis was completed using Cogent NGS Immune Viewer software.